Sinomenium acutum Modulates Platelet Aggregation and Thrombus Formation by Regulating the Glycoprotein VI-Mediated Signalosome in Mice

Sinomenium acutum (SA) has long been used as a traditional medicine in China, Japan, and Korea to treat a wide range of diseases. It has been traditionally used to ameliorate inflammation and improve blood circulation. However, its role in platelet activation has not been thoroughly investigated. Hence, we conducted this study to assess the potential inhibitory effect of SA on platelet aggregation and thrombus formation. The antiplatelet activities of SA were evaluated by assessing platelet aggregation, granular secretion, intracellular Ca2+ mobilization, and the Glycoprotein (GP) VI-mediated signalosome. The thrombosis and bleeding time assays were used to investigate the effect of SA (orally administered at 50 and 100 mg/kg for seven days) in mice. SA treatment at concentrations of 50, 100, and 200 μg/mL significantly reduced GPVI-mediated platelet aggregation, granular secretion, and intracellular Ca2+ mobilization. Further biochemical studies revealed that SA inhibited spleen tyrosine kinase, phospholipase Cγ2, phosphatidylinositol 3-kinase, and AKT phosphorylation. Interestingly, oral administration of SA efficiently ameliorated FeCl3-induced arterial thrombus formation without prolonging the tail bleeding time. These findings suggest that SA has beneficial effects in thrombosis and hemostasis. Therefore, SA holds promise as an effective therapeutic agent for the treatment of thrombotic diseases.


Introduction
Platelets are small, anucleate cell fragments that are essential for blood clotting and preventing bleeding from injuries.Unwanted platelet activation, however, is a significant contributor to the development and progression of cardiovascular disease.Once activated, platelets rapidly adhere to immobilized adhesive proteins such as von Willebrand factor and collagen, triggering platelet activation and aggregation, leading to the formation of platelet-rich thrombi.These thrombi can obstruct arteries and reduce arterial blood flow, thereby causing various diseases, including hypertension, lower-extremity deep venous thrombosis, atrial fibrillation, infective endocarditis, and heart failure [1,2].
Glycoprotein VI (GPVI) is a crucial platelet surface receptor involved in platelet function and activation.When platelets come into contact with exposed collagen, which is found in damaged blood vessel walls, a signaling cascade is initiated, resulting in platelet activation and aggregation.GPVI binds to collagen, recruits, and associates with another platelet receptor known as Fc receptor γ-chain [3].The FcRγ-chain is essential for GPVI signaling and is also required for the expression of GPVI on the platelet surface.When Pharmaceuticals 2024, 17, 6 2 of 13 GPVI forms a dimer, Src family kinases such as Fyn and Lyn phosphorylate the tyrosine residue of the FcRγ on its motif.This subsequently initiates a spleen tyrosine kinase (Syk)-dependent signaling cascade, leading to the recruitment of the linker of activated T cells (LAT) signalosome involving phospholipase C (PLC) γ2 and phosphoinositide-3 kinase (PI3K) [4][5][6].The tyrosine phosphorylation-based activation of PLCγ2 eventually leads to intracellular Ca 2+ accumulation, resulting in platelet activation, the release of granules, shape change, and the formation of platelet aggregates [7,8].The activation of GPVI-mediated platelet function also triggers the release of various soluble factors, such as adenosine diphosphate (ADP) and thromboxane A2, which further enhance platelet activation and aggregation [3].Dysfunction or abnormalities in GPVI signaling profoundly affect platelet function and hemostasis.For instance, defects in GPVI signaling pathways can lead to bleeding disorders, while excessive or inappropriate GPVI activation can contribute to thrombotic conditions such as arterial thrombosis [9].Ongoing research on GPVI and its role in platelet function has identified the GPVI signaling pathway as a pivotal focus in the development of antiplatelet drugs.
Sinomenium acutum (Thumb.)Rehd.et Wils.(Menispermaceae, SA) has long been used as a traditional medicine in China, Japan, and Korea for the treatment of various diseases.It contains an alkaloid called sinomenine, which has received significant research attention for its potential anti-inflammatory, immunosuppressive, and analgesic properties [10,11].As a result, SA has been used to alleviate pain and reduce inflammation associated with conditions like arthritis, rheumatism, and joint disorders.Furthermore, it has been used to treat fever, skin rashes, and digestive disorders [10,11].However, the scientific information on SA remains unclear.Therefore, we conducted this study to evaluate the antiplatelet effect of SA on the process of thrombotic diseases using platelet aggregation and plateletthrombus formation models.
The present study has demonstrated that the ethanol extract of SA has the ability to inhibit thrombus formation in vivo and in vitro, particularly by inhibiting platelet activation and aggregation induced by collagen and collagen-related peptide (CRP).In a mouse model of ferric chloride (FeCl 3 )-induced thrombosis, SA has been found to play a crucial role in collagen-induced platelet-thrombus formation.Notably, the administration of SA did not significantly increase the tail bleeding time compared to the control mice.These findings highlight the potential of SA to exert antiplatelet and antithrombotic effects without affecting hemostasis.Hence, SA holds great promise as an effective therapeutic agent for the treatment of thrombotic diseases.

SA Suppresses GPVI-Activated Platelet Aggregation and ATP Secretion
To investigate the role of SA in regulating platelet function, platelet aggregation was first assessed.Our findings revealed that stimulation with various agonists, including collagen (1 µg/mL), CRP (0.5 µg/mL), thrombin (0.05 U/mL), U46619 (3 µM), and ADP (2.5 µM), significantly elevated platelet aggregation (Figure 1).However, compared to the vehicle control, SA-treated platelets showed less aggregation stimulated by collagen (≤1 µg/mL) and CRP (≤0.5 µg/mL) (Figure 1A,B).In contrast, thrombin (≤0.05 U/mL), U46619 (≤3 µM), and ADP (≤2.5 µM) did not elicit such an effect (Figure 1C-E).Furthermore, higher concentrations of collagen (≥3 µg/mL) and CRP (≥2 µg/mL) invalidated the observed defects in SA-treated platelets (Figure S1).To further estimate the effect of SA on platelet function, ATP secretion was assessed.Our findings demonstrated that SA treatment at concentrations of 50, 100, and 200 µg/mL caused a significant decrease in ATP secretion facilitated by collagen (≤1 µg/mL) and CRP (≤0.5 µg/mL) compared to the vehicle control (Figure 1A,B).However, stimulation with thrombin (0.05 U/mL) or U46619 (3 µM) did not yield comparable results (Figure 1C,D).The effect of SA on platelet activation using PRP was also investigated in comparison with that in washed platelets.We observed that collagen (1 µg/mL)or CRP (1 µg/mL)-stimulated platelet aggregation in PRP was diminished by SA in a concentration-dependent manner similar to the effects of washing platelets (Figure 1F,G).These results suggest that plasma proteins do not influence the effects of SA on platelet aggregation.These findings suggest that SA plays an important role in GPVI-mediated platelet aggregation and ATP secretion.that collagen (1 µg/mL)-or CRP (1 µg/mL)-stimulated platelet aggregation in PRP was diminished by SA in a concentration-dependent manner similar to the effects of washing platelets (Figure 1F,G).These results suggest that plasma proteins do not influence the effects of SA on platelet aggregation.These findings suggest that SA plays an important role in GPVI-mediated platelet aggregation and ATP secretion.In ATP secretion, cleansed platelets' surfaces were prepared with the various concentrations of SA (50, 100, and 200 µg/mL) for 10 min at 37 °C.After the addition of the luciferin/luciferase reagent, platelets were activated with various agonists, then ATP secretion was assessed with a luminometer.PRP was preincubated with either 0.01% DMSO, or one of three concentrations of SA (50, 100, or 200 µg/mL) for 10 min at 37 °C.They were then stimulated with 1 µg/mL collagen (F) or 0.5 µg/mL CRP  To evaluate the effects of SA on platelet activation, P-selectin exposure and αIIbβ3 integrin activation, which are key processes in the positive feedback cycle of platelet activation, were assessed.In a dose-dependent manner, SA at concentrations of 50, 100, and 200 µg/mL significantly reduced P-selectin exposure and lowered αIIbβ3 integrin activation upon CRP (0.5 µg/mL) stimulation (Figure 2A,B).These findings indicate that SA effectively inhibits GPVI-mediated platelet granular secretion and αIIbβ3 integrin activation.Next, we examined the mechanism explaining the association between SA treatment and platelet activation, along with the GPVI-stimulated increase in intracellular Ca 2+ .Increases in intracellular Ca 2+ arise through either Ca 2+ release from intracellular Ca 2+ stores or influx across the plasma membrane [12].Our findings demonstrated a significant increase in intracellular Ca 2+ release and inflow upon stimulation with CRP (0.5 µg/mL).Moreover, when platelets were pretreated with SA (50, 100, and 200 µg/mL) prior to CRP stimulation, Ca 2+ mobilization was inhibited in a dose-dependent manner (Figure 2C).These findings indicate that SA can modulate CRP-triggered intracellular Ca 2+ release and influx.

SA Is Actively Involved in P-Selectin Exposure, αIIbβ3 Integrin Activation, and Ca 2+ Mobilization after CRP Induction
To evaluate the effects of SA on platelet activation, P-selectin exposure and αIIbβ3 integrin activation, which are key processes in the positive feedback cycle of platelet activation, were assessed.In a dose-dependent manner, SA at concentrations of 50, 100, and 200 µg/mL significantly reduced P-selectin exposure and lowered αIIbβ3 integrin activation upon CRP (0.5 µg/mL) stimulation (Figure 2A,B).These findings indicate that SA effectively inhibits GPVI-mediated platelet granular secretion and αIIbβ3 integrin activation.Next, we examined the mechanism explaining the association between SA treatment and platelet activation, along with the GPVI-stimulated increase in intracellular Ca 2+ .Increases in intracellular Ca 2+ arise through either Ca 2+ release from intracellular Ca 2+ stores or influx across the plasma membrane [12].Our findings demonstrated a significant increase in intracellular Ca 2+ release and inflow upon stimulation with CRP (0.5 µg/mL).Moreover, when platelets were pretreated with SA (50, 100, and 200 µg/mL) prior to CRP stimulation, Ca 2+ mobilization was inhibited in a dose-dependent manner (Figure 2C).These findings indicate that SA can modulate CRP-triggered intracellular Ca 2+ release and influx.The preventive effect of Sinomenium acutum (SA) on P-selectin exposure, αIIbβ3 integrin stimulation, and Ca 2+ mobilization.The binding of anti-P-selectin (A) and anti-activated αIIbβ3 (JON/A) antibodies (B) to platelets was measured as the ratio of the geometric mean fluorescence intensity value of antibodies to that of the control immunoglobulin G.The quantitative data are presented as means ± standard deviations (n = 3).In a Ca 2+ mobilization assay, mouse platelets were incubated with a calcium-sensitive dye for 30 min at 37 • C in the dark, and then platelets were induced using 0.5 µg/mL collagen-related peptide (CRP) for 10 min.Intracellular Ca 2+ secretion and flow (C) were evaluated and quantified with the area under the curve (arbitrary units).The quantitative data are demonstrated as means ± standard deviations (n = 4).*, p < 0.05; **, p < 0.01; and ***, p < 0.001.The reaction to GPVI-specific agonists involves a substantial enhancement of Ca 2+ signaling through the phosphorylation of Syk, PLCγ2, and PI3K signaling molecules [13], which, in turn, monitors platelet activation.Since SA treatment is important for the following Ca 2+ mobilization, the effect of SA treatment on regulating Syk, PLCγ2, and PI3K phosphorylation was explored.Our findings demonstrate that SA treatment, compared to the control group, significantly diminished the phosphorylation of Syk, PLCγ2, and PI3K, as well as the downstream kinase AKT and mitogen-activated protein kinases (MAPKs), following CRP induction (Figure 3).These findings indicate that SA plays an essential role in regulating Syk, PLCγ2, PI3K, AKT, and extracellular signal-regulated kinase (ERK) phosphorylation without displaying selectivity toward specific signal transduction pathways.induced using 0.5 µg/mL collagen-related peptide (CRP) for 10 min.Intracellular Ca 2+ secretion and flow (C) were evaluated and quantified with the area under the curve (arbitrary units).The quantitative data are demonstrated as means ± standard deviations (n = 4).*, p < 0.05; **, p < 0.01; and ***, p < 0.001.

SA Is Essential in Regulating Syk, PLCγ2, PI3K, AKT, and ERK Phosphorylation following CRP Activation
The reaction to GPVI-specific agonists involves a substantial enhancement of Ca 2+ signaling through the phosphorylation of Syk, PLCγ2, and PI3K signaling molecules [13], which, in turn, monitors platelet activation.Since SA treatment is important for the following Ca 2+ mobilization, the effect of SA treatment on regulating Syk, PLCγ2, and PI3K phosphorylation was explored.Our findings demonstrate that SA treatment, compared to the control group, significantly diminished the phosphorylation of Syk, PLCγ2, and PI3K, as well as the downstream kinase AKT and mitogen-activated protein kinases (MAPKs), following CRP induction (Figure 3).These findings indicate that SA plays an essential role in regulating Syk, PLCγ2, PI3K, AKT, and extracellular signal-regulated kinase (ERK) phosphorylation without displaying selectivity toward specific signal transduction pathways.

SA prevents In Vivo Thrombosis, While Having No Impact on Hemostasis
To explore the influence of SA on arterial thrombus generation, a mouse model of FeCl3-promoted carotid artery thrombosis was developed.Thrombus formation was assessed using a 10% (460 mM) FeCl3 solution.After FeCl3 application, carotid occlusion was observed at a mean time of 9.73 ± 2.23 min in the control group.However, SA therapy increased the carotid occlusion time to 15.78 ± 2.72 min at a concentration of 50 mg/kg BW or 20.83 ± 2.64 min at a concentration of 100 mg/kg BW compared to that in the positive control (27.98 ± 2.91 min) at an ASA concentration of 100 mg/kg BW (Figure 4A,B).To further evaluate the impact of oral SA administration on hemostatic function, the tail

SA prevents In Vivo Thrombosis, While Having No Impact on Hemostasis
To explore the influence of SA on arterial thrombus generation, a mouse model of FeCl 3 -promoted carotid artery thrombosis was developed.Thrombus formation was assessed using a 10% (460 mM) FeCl 3 solution.After FeCl 3 application, carotid occlusion was observed at a mean time of 9.73 ± 2.23 min in the control group.However, SA therapy increased the carotid occlusion time to 15.78 ± 2.72 min at a concentration of 50 mg/kg BW or 20.83 ± 2.64 min at a concentration of 100 mg/kg BW compared to that in the positive control (27.98 ± 2.91 min) at an ASA concentration of 100 mg/kg BW (Figure 4A,B).To further evaluate the impact of oral SA administration on hemostatic function, the tail bleeding time was assessed as the timepoint of ceased bleeding after tail amputation.Our findings demonstrated no statistically significant differences in the value of this parameter between two studied groups (Figure 4C).Furthermore, the volume of blood collected from an amputation site, stratified according to hemoglobin content, did not differ between the groups.However, oral administration of ASA at a dose of 100 mg/kg significantly elevated the bleeding time and increased hemoglobin levels (Figure 4D).These findings indicate that SA plays an important role in arterial thrombosis in vivo, while not interfering with the processes of hemostasis.
bleeding time was assessed as the timepoint of ceased bleeding after tail amputation.Our findings demonstrated no statistically significant differences in the value of this parameter between two studied groups (Figure 4C).Furthermore, the volume of blood collected from an amputation site, stratified according to hemoglobin content, did not differ between the groups.However, oral administration of ASA at a dose of 100 mg/kg significantly elevated the bleeding time and increased hemoglobin levels (Figure 4D).These findings indicate that SA plays an important role in arterial thrombosis in vivo, while not interfering with the processes of hemostasis.After oral administration of 0.5% low-viscosity carboxymethylcellulose (CMC) and/or SA (50 and 100 mg/kg, body weight) or acetylsalicylic acid (ASA; 100 mg/kg, body weight) once a day for seven days (A,B), 10% FeCl3 was injected into the mouse carotid artery for two minutes, and blood flow traces were controlled until stable occlusion was achieved.In the bleeding time assay, the tails of mice were removed, and the bleeding time (C) was monitored, as demonstrated in Methods.(D) Blood loss during the bleeding time assay was measured by assessing the absorbance at 575 nm for hemoglobin (Hb).The horizontal bars illustrate the median occlusion time (n = 10).# , p < 0.05, ## , p < 0.01 and ### , p < 0.001 versus the vehicle control after Student's t-test.

Phytochemical Profiling of SA
UHPLC-MS/MS analysis is widely utilized because it has high sensitivity and resolution and facilitates the systematic profiling of active components presents within plants [14,15].In this study, Sinomenium acutum extract was analyzed via UHPLC-MS/MS to identify major components.Through a comparison of the retention time (Rt) and mass spectra with reference standards, we identified eleven phytochemicals predominantly present in the extract.These include higenamine, acutumidine, acutumine, sinomenine, gelsemine, magnoflorine, scopoletin, columbamine (or jatrorrhizine), palmatine, syringin, and eleutheroside E, all of which are found in Sinomenium acutum (Table 1).After oral administration of 0.5% low-viscosity carboxymethylcellulose (CMC) and/or SA (50 and 100 mg/kg, body weight) or acetylsalicylic acid (ASA; 100 mg/kg, body weight) once a day for seven days (A,B), 10% FeCl 3 was injected into the mouse carotid artery for two minutes, and blood flow traces were controlled until stable occlusion was achieved.In the bleeding time assay, the tails of mice were removed, and the bleeding time (C) was monitored, as demonstrated in Methods.(D) Blood loss during the bleeding time assay was measured by assessing the absorbance at 575 nm for hemoglobin (Hb).The horizontal bars illustrate the median occlusion time (n = 10).# , p < 0.05, ## , p < 0.01 and ### , p < 0.001 versus the vehicle control after Student's t-test.

Phytochemical Profiling of SA
UHPLC-MS/MS analysis is widely utilized because it has high sensitivity and resolution and facilitates the systematic profiling of active components presents within plants [14,15].In this study, Sinomenium acutum extract was analyzed via UHPLC-MS/MS to identify major components.Through a comparison of the retention time (Rt) and mass spectra with reference standards, we identified eleven phytochemicals predominantly present in the extract.These include higenamine, acutumidine, acutumine, sinomenine, gelsemine, magnoflorine, scopoletin, columbamine (or jatrorrhizine), palmatine, syringin, and eleutheroside E, all of which are found in Sinomenium acutum (Table 1).Figure 5 illustrates the base peak chromatograms and the extracted ion chromatograms of these identified phytochemicals in Sinomenium acutum.

Discussion
For this study, we evaluated the effects of SA ethanol extract on platelet function including antithrombotic effects.Our findings revealed that SA exhibited a protective effect

Discussion
For this study, we evaluated the effects of SA ethanol extract on platelet function including antithrombotic effects.Our findings revealed that SA exhibited a protective effect against platelet aggregation and activation and improved platelet-thrombus formation via the GPVI-mediated platelet signalosome.Previous studies have primarily focused on the antioxidant, anti-inflammatory, analgesic, anti-allergic, immunosuppressive, antitumor, liver-protective, and other effects of SA.Clinical use of SA therapy has mainly been documented in relation to rheumatoid arthritis, ankylosing spondylitis, and other diseases [10,11].However, this study is the first to describe the antiplatelet effect of SA on GPVI-mediated platelet aggregation and thrombus formation.
To investigate the underlying mechanism, we investigated GPVI-mediated intracellular Ca 2+ mobilization, granular secretion, and fibrinogen binding to integrin αIIbβ3 (JON/A).Our findings demonstrated that SA extract markedly inhibited intracellular Ca 2+ activation, ATP secretion, P-selectin exposure, and integrin αIIbβ3 stimulation.Targeting the collagen receptor GPVI has been shown to be an effective approach to reducing thrombosis while maintaining hemostasis [18].Furthermore, clinical trials investigating the humanized anti-GPVI Fab fragment (ACT01) and the dimeric GPVI-Fc fusion protein (Revacept) demonstrated their inhibitory effects on the interaction between platelets and collagen without affecting general hemostasis [19,20].These findings suggest that targeting GPVI could be a promising approach for antithrombotic treatment.In the present study, both the biochemical and in vivo results demonstrated that SA treatment could effectively prevent GPVI-mediated platelet aggregation and thrombus formation without increasing the risk of bleeding.The inhibitory effects of SA extract on platelet aggregation, activation, degranulation, and thrombus formation might be based on the inhibitory effects of the GPVI-mediated signaling pathway during cell activation.
The binding of the GPVI to immobilized collagen initiates the adhesion of flowing platelets to the subendothelial extracellular matrix and, thereby, enables the signaling pathway, which is initiated by the Src-family-kinase-mediated phosphorylation of tyrosine residues associated with the immunoreceptor tyrosine-based activation motif containing FcRγ chains [3].Subsequently, phospholipase Cγ2 (PLCγ2) is activated by the signaling cascades involved in the recruitment and activation of spleen tyrosine kinase (Syk), the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), Proto-oncogene vav (Vav1), phosphatidylinositol 3-kinase (PI3K), and Bruton's tyrosine kinase (Btk).The tyrosine phosphorylation-based activation of PLCγ2 eventually leads to intracellular Ca 2+ accumulation, a marker of platelet activation and thrombus formation [7,8].We observed that SA extract significantly inhibited the increase in phosphorylation of Syk, PLCγ2, PI3K, and AKT induced by CRP [21][22][23].It has been demonstrated that the phosphorylation of PI3K and AKT is highly expressed in platelets, and these are key signaling pathways for GPVI downstream activation induced by collagen [24][25][26].The PI3K and AKT pathways are downstream of Src family kinases and are the integral part of platelet activation by influencing intracellular Ca 2+ mobilization, granular secretion, and platelet aggregation [25][26][27].Thus, our findings suggest that the inhibitory effects of SA extract on the phosphorylation of Syk, PLCγ2, PI3K, and AKT pathways comprise an efficient and safe antiplatelet and antithrombotic strategy for GPVI-mediated platelet functions.
The FeCl 3 -promoted vascular injury model is commonly used to assess antithrombotic activity and has been well established for evaluating in vivo antithrombotic effects [2].Tseng et al. demonstrated that FeCl 3 accesses the endothelium through small vesicles via an endocytic-exocytic route, resulting in complete endothelial denudation [28].This process exposes the subendothelial matrix (collagen) and leads to arterial thrombus formation with platelet activation and fibrin formation, which can be attenuated using antiplatelet agents [29].In our study, we found that SA treatment significantly increased vascular occlusion times in mice by suppressing platelet activation without expanding the bleeding time compared to vehicle treatment.In addition, because FeCl 3 -induced injury can disrupt the endothelium, our findings further suggest that SA is important for regulating endothelial cells in arterial thrombosis.Interestingly, SA treatment did not affect the hemostatic function at the site of tail transaction [30].Noting that the tail bleeding time may not be a reliable analysis of platelet contribution to hemostatic function, we also observed no increased bleeding from the surgery site during the FeCl 3 -induced injury arterial thrombosis study.Additionally, depending on the activities and concentrations of herbal extracts, results might differ [31].We also observed that SA did not differ in the coagulation parameter compared with the control group (Supplemental Table S1).Consequently, unless SA concentrations exceed threshold concentration levels, there is still a chance that SA treatment will result in a reduction in antiplatelets without anticoagulation properties.Therefore, our findings suggest that SA is a potent natural candidate for alleviating platelet-related cardiovascular diseases.

Flow Cytometric Analysis
The rinsed platelets were treated with either 0.01% DMSO or one of three concentrations of SA (50, 100, or 200 µg/mL) for 10 min at 37 • C.They were then processed with 0.5 µg/mL of CRP for five minutes at 37 • C and incubated with phycoerythrin-conjugated antibodies against P-selectin or induced αIIbβ3 integrin (JON/A) for 15 min.The cells were then evaluated using flow cytometry (Gallios, Beckman Coulter, Bera, CA, USA).

Ca 2+ Mobilization
The rinsed platelets (1 × 10 8 /mL) were immersed in HEPES-Tyrode buffer (pH 7.4) without CaC l2 and subjected to treatment with either 0.01% DMSO or one of three concentrations of SA (50, 100, or 200 µg/mL) for 10 min at 37 • C. The cells were then stained with a Ca 2+ dye (FLIPR Calcium 5 Assay kit) for 30 min at 37 • C in the dark and then stimulated with CRP (0.2 µg/mL).Cytosolic Ca 2+ levels were determined with a spectrofluorometer (Spectramax I3, Molecular Devices) with an excitation wavelength of 485 nm and an emission of 525 nm.Ca 2+ mobilization was evaluated by measuring the area under the curve and expressed as a relative fluorescence unit.

Immunoblotting
Mouse platelets were preincubated with three different concentrations of SA (50, 100, or 200 µg/mL) for 10 min and then stimulated with 0.5 µg/mL CRP for 5 min under constant stirring at 1000 rpm in an aggregometer.To measure the phosphorylation levels of kinases, platelets were lysed in lysis buffer.The lysed platelets were then sonicated.An equal amount of protein (30 µg) was electrophoresed under reduced conditions and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA).The immunoblot was blocked with TBS-T buffer containing 5% BSA for 1 h and incubated overnight at 4 • C with different primary antibodies.After being washed 4 times for 10 min each with TBS-T buffer, the blots were probed with a Horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Santa CruZ Biotechnology, Santa Cruz, CA, USA, sc-2004 and sc-2005) for 1 h at room temperature, and the membranes were visualized using enhanced chemiluminescence.The band density was quantified via densitometry using ImageJ software (v1.52a).Phosphorylation levels of kinases were determined by normalizing the density of antibodies against phosphorylated kinases to that of antibodies against total kinases.

Figure 2 .
Figure 2.The preventive effect of Sinomenium acutum (SA) on P-selectin exposure, αIIbβ3 integrin stimulation, and Ca 2+ mobilization.The binding of anti-P-selectin (A) and anti-activated αIIbβ3 (JON/A) antibodies (B) to platelets was measured as the ratio of the geometric mean fluorescence intensity value of antibodies to that of the control immunoglobulin G.The quantitative data are presented as means ± standard deviations (n = 3).In a Ca 2+ mobilization assay, mouse platelets were incubated with a calcium-sensitive dye for 30 min at 37 °C in the dark, and then platelets were

Figure 2 .
Figure 2.The preventive effect of Sinomenium acutum (SA) on P-selectin exposure, αIIbβ3 integrin stimulation, and Ca 2+ mobilization.The binding of anti-P-selectin (A) and anti-activated αIIbβ3 (JON/A) antibodies (B) to platelets was measured as the ratio of the geometric mean fluorescence intensity value of antibodies to that of the control immunoglobulin G.The quantitative data are presented as means ± standard deviations (n = 3).In a Ca 2+ mobilization assay, mouse platelets were incubated with a calcium-sensitive dye for 30 min at 37 • C in the dark, and then platelets were induced using 0.5 µg/mL collagen-related peptide (CRP) for 10 min.Intracellular Ca 2+ secretion and flow (C) were evaluated and quantified with the area under the curve (arbitrary units).The quantitative data are demonstrated as means ± standard deviations (n = 4).*, p < 0.05; **, p < 0.01; and ***, p < 0.001.

Figure 4 .
Figure 4. Sinomenium acutum (SA) delays FeCl3-induced arterial thrombus formation without causing bleeding.FeCl3-activated arterial thrombus formation was carried out, as shown in Methods.After oral administration of 0.5% low-viscosity carboxymethylcellulose (CMC) and/or SA (50 and 100 mg/kg, body weight) or acetylsalicylic acid (ASA; 100 mg/kg, body weight) once a day for seven days (A,B), 10% FeCl3 was injected into the mouse carotid artery for two minutes, and blood flow traces were controlled until stable occlusion was achieved.In the bleeding time assay, the tails of mice were removed, and the bleeding time (C) was monitored, as demonstrated in Methods.(D) Blood loss during the bleeding time assay was measured by assessing the absorbance at 575 nm for hemoglobin (Hb).The horizontal bars illustrate the median occlusion time (n = 10).# , p < 0.05, ## , p < 0.01 and ### , p < 0.001 versus the vehicle control after Student's t-test.

Figure 4 .
Figure 4. Sinomenium acutum (SA) delays FeCl 3 -induced arterial thrombus formation without causing bleeding.FeCl 3 -activated arterial thrombus formation was carried out, as shown in Methods.After oral administration of 0.5% low-viscosity carboxymethylcellulose (CMC) and/or SA (50 and 100 mg/kg, body weight) or acetylsalicylic acid (ASA; 100 mg/kg, body weight) once a day for seven days (A,B), 10% FeCl 3 was injected into the mouse carotid artery for two minutes, and blood flow traces were controlled until stable occlusion was achieved.In the bleeding time assay, the tails of mice were removed, and the bleeding time (C) was monitored, as demonstrated in Methods.(D) Blood loss during the bleeding time assay was measured by assessing the absorbance at 575 nm for hemoglobin (Hb).The horizontal bars illustrate the median occlusion time (n = 10).# , p < 0.05, ## , p < 0.01 and ### , p < 0.001 versus the vehicle control after Student's t-test.
*, The comparison with the reference values of retention time and mass spectrum.R t , retention time.

Table 1 .
Phytochemical components of Sinomenium acutum via UHPLC-MS/MS., The comparison with the reference values of retention time and mass spectrum.Rt, retention time. *